Metabolic fingerprinting of rat urine by LC/MS Part 1. Analysis by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry

Complex biological samples, such as urine, contain a very large number of endogenous metabolites reflecting the metabolic state of an organism. Metabolite patterns can provide a comprehensive signature of the physiological state of an organism as well as insights into specific biochemical processes. Although the metabolites excreted in urine are commonly highly polar, the samples are generally analyzed using reversed-phase liquid chromatography mass spectrometry (RP-LC/MS). In Part 1 of this work, a method for detecting highly polar metabolites by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) is described as a complement to RP-LC/ESI-MS. In addition, in an accompanying paper (Part 2), different multivariate approaches to extracting information from the resulting complex data are described to enable metabolic fingerprints to be obtained. The coverage of the method for the screening of as many metabolites as possible is highly improved by analyzing the urine samples using both a C(18) column and a ZIC-HILIC column. The latter was found to be a good alternative when analyzing highly polar compounds, e.g., hydroxyproline and creatinine, to columns typically used for reversed-phase liquid chromatography.     

Metabolic fingerprinting of rat urine by LC/MS Part 2. Data pretreatment methods for handling of complex data

Metabolic fingerprinting of biofluids like urine is a useful technique for detecting differences between individuals. With this approach, it might be possible to classify samples according to their biological relevance. In Part 1 of this work a method for the comprehensive screening of metabolites was described, using two different liquid chromatography (LC) column set-ups and detection by electrospray ionization mass spectrometry (ESI-MS). Data pretreatment of the resulting data described in is needed to reduce the complexity of the data and to obtain useful metabolic fingerprints. Three different approaches, i.e., reduced dimensionality (RD), MarkerLynx, and MS Resolver, were compared for the extraction of information. The pretreated data were then subjected to multivariate data analysis by partial least squares discriminant analysis (PLS-DA) for classification. By combining two different chromatographic procedures and data analysis, the detection of metabolites was enhanced as well as the finding of metabolic fingerprints that govern classification. Additional potential biomarkers or xenobiotic metabolites were detected in the fraction containing highly polar compounds that are normally discarded when using reversed-phase liquid chromatography.     

Evaluation of an on-line solid-phase extraction method for determination of almokalant, an antiarrhythmic drug, by liquid chromatography

A fully automated liquid chromatographic method based on a Prospekt solid-phase extraction unit is described for determination of the antiarrhythmic drug almokalant in plasma. The assay comprises solid-phase extraction on a C₂ phase and separation on a C₁₈ column with fluorometric detection. In the original procedure 40 samples a day could be run unattended but by modifying the sequence in the solid-phase extraction process it was possible to increase this number to 70. The method gives an absolute recovery of 92% and a repeatability (C.V.) of 2.9% at 75 nmol/1 of plasma. The limit of quantitation is 2 nmol/1 of plasma (C.V. < 20%). As regards accuracy and precision the performance of the method is as good as the manual method based on liquid-liquid extraction. The Prospekt method is, above all, faster and requires far less manual effort.     

Comparison of the effect of the linseed extract Salinum® and a methyl cellulose preparation on the symptoms of dry mouth

The effect of a linseed extract Salinum® and a sodium carboxymethyl cellulose preparation called MAS-84 was compared with regard to its effect on the symptoms of dry mouth. Twenty patients with xerostomia, who had been treated for cancer in the head and neck by radiation were recruited from the clinic for maxillofacial surgery, Malmö University Hospital. Following radiation treatment the salivation was severely reduced. The symptoms of a general feeling of a dry mouth, difficulties in chewing and swallowing, taste disturbances, problems with speech and mouth burning were registered on a subjective verbal rating scale. In addition plaque index and gingival bleeding index were determined. The study design was crossover and performed single blind. The experimental period was 7 weeks. The patients were randomly divided into 2 groups, One group used Salinum® and the other MAS-84 for 3 weeks. The fourth week was a wash out period and for the next three weeks the patients shifted preparation. Each of the preparations was used ad libitium. Registrations of the various parameters were undertaken on days 0, 7 and 21 of the respective period. At the initial examination all patients reported considerable disturbances from mouth-dryness. These symptoms were reduced in 15 patients during the Salinum® period and in 9 during the MAS-84 period. The relief was significantly more pronounced during the use of Salinum® compared to that during the use of the methyl cellulose preparation. On day 21 plaque and gingival bleeding were significantly reduced during the Salinum® period but not during the MAS-84 period. The results of the present study confirm those of a previous pilot study and indicate that the linseed mucilage significantly reduced the symptoms of dry mouth. This effect increased with increasing time of saliva substitute use. The linseed mucilage Salinum® appeared to be a suitable saliva replacement in mouth dry patients.     

Diphosphoinositol pentakisphosphate as a novel mediator of insulin exocytosis

The pancreatic β-cell has served as an important model system for the revelation of new physiological roles for inositides. Initially, our studies were restricted to the role of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) in the regulation of cytoplasmic free calcium concentration ([Ca²⁺]_i), but it soon became clear that other inositol phosphates could also regulate β-cell [Ca²⁺]_i. For example, inositol hexakisphosphate (InsP₆) promotes the opening of the key voltage-dependent L-type Ca²⁺ channels, responsible for Ca²⁺ influx and the final release of insulin. Furthermore, InsP₆ and inositol lipids such as phosphatidylinositol 4,5-bisphosphate are intimately involved the regulation of endocytosis and exocytosis. We now review our most recent work, which has focused on the phosphorylation product of InsP₆, diphosphoinositol pentakisphosphate (PP-InsP₅ or InsP₇). We have established that InsP₇ via the activity of the InsP₆ kinase, IP6K1, promotes insulin release from the readily releasable pool of vesicles (RRP). The RRP is thought to be synonymous with the first phase of insulin secretion. This has a direct implication for type 2 diabetes, as it is this initial phase of insulin release that is curtailed in the disease. Hints from human genetic linkage studies suggest that disruption of IP6K1 could be a factor in the development of type 2 diabetes and a recent mouse model, where IP6K1 is universally deleted, exhibits lowered plasma insulin levels. Hence, this is yet another important example demonstrating how the β-cell utilizes inositides in the regulation of function. Although we do not fully understand the underlying molecular mechanisms, it is clear that IP6K1-mediated production of InsP₇ has an essential role in the regulation of the insulin secretory process.     

Resveratrol prevents dexamethasone-induced expression of the muscle atrophy-related ubiquitin ligases atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent mechanism

Resveratrol (3,5,4'-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.     

Munc18-1 and Munc18-2 proteins modulate beta-cell Ca2+ sensitivity and kinetics of insulin exocytosis differently

Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in β-cell stimulus-secretion coupling. We show that pancreatic β-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. Ramp-like photorelease of caged Ca(2+) and high resolution whole-cell patch clamp recordings show that Munc18-1 and Munc18-2 overexpression shift the Ca(2+) sensitivity of the fastest phase of insulin exocytosis differently. In addition, we reveal that Ca(2+) sensitivity of exocytosis in β-cells depends on the phosphorylation status of the Munc18 proteins. Even though Munc18-1 emerges as the key SM-protein determining the Ca(2+) threshold for triggering secretory activity in a stimulated β-cell, Munc18-2 has the ability to increase Ca(2+) sensitivity and thus mediates the release of fusion-competent granules requiring a lower cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i)(.) Hence, Munc18-1 and Munc18-2 display distinct subcellular compartmentalization and can coordinate the insulin exocytotic process differently as a consequence of the actual [Ca(2+)](i).     

Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming beta cell function in humans

Acetylcholine is a neurotransmitter that has a major role in the function of the insulin-secreting pancreatic beta cell. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to the case in mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of human islets provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. Cholinergic signaling within islets represents a potential therapeutic target in diabetes, highlighting the relevance of this advance to future drug development.